Tuesday, April 30, 2013

Dual luciferase assays have become the standard assay when
information on the effect of regulatory elements and proteins with regard to
regulation of gene expression is desired. Dual luciferase assays use one
reporter to measure the test conditions while the second serves as a control
for transfection efficiency and cell viability. Typically this has employed
sequential activation of test and control with requisite quenching.

The use of spectrally resolved forms of luciferase allow for
the detection of two luciferases in one sample simultaneously without
quenching. The POLARstar Omega from BMG LABTECH is ideally suited for
monitoring activity from two luciferases in a spectrally resolved manner. The
performance of the POLARstar Omega using the Thermo Scientific Pierce Dual
Spectral Luciferase assay is described in application note 233

Dual Spectral Luciferase assays employ spectrally resolved
pairs of luciferase that can be detected in a single sample with high
sensitivity in a simple one-step protocol. The simultaneous dual emission
detection capacity of the POLARstar Omega further allows the detection to be
performed with a single read.

Unlike the traditional dual luciferase assays which measure luciferase sequentially and require quenching of the first signal so that the second signal may be detected, the use of spectrally resolved luciferases allows for simplified one step detection. By pairing red firefly luciferase with green Renilla, Gaussia or Cypridina luciferase and selecting appropriate filters the activity of both luciferase enzymes can be detected in one sample. As a result you will be able to perform gene expression analysis in a simplified platform which is made even easier using the simultaneous dual emission detection capability of the POLARstar Omega.

Thursday, April 25, 2013

Dual luciferase reporters have become the go to assay to assess changes in gene expression that result from altering the activity of components of a biological pathway. The standard approach employs sequential detection of one luciferase that relates to the studied subject and a second luciferase that is used to indicate cell viability and transfection efficiency.

Selecting filter pairs that allowed for sensitive detection of each luciferase pair while limiting the interference between them was key to the success of this application. Application note 233 clearly describes the final filter sets employed that allow for the accurate measurement of each reporter over a 100,000-fold concentration range for three possible combinations of spectrally resolved luciferases.

The study confirms the effect of heat on carbon efflux, such that higher temperatures lead to higher carbon efflux as a result of all organisms increasing respiration in response to increased heat.The surprise was that urban lawns had a higher carbon efflux than did agricultural areas. However, the authors point out that urban areas are known to be exposed to higher heat. These so called heat islands that result from the dark surfaces; black top, roofs, etc.; that characterize urban development. They propose that these heat islands have more localized effects leading to increased soil temperatures for residential lawns and thus the observed higher carbon output. Dr. Bowne intends to continue research in this area in hopes of uncovering how changes in land use affect the carbon cycle.

Friday, April 19, 2013

As most if not all of you we at BMG LABTECH are watching the events in Boston closely. We are of course concerned for the citizens of the area and hope that the apparent instigators of the attack are apprehended quickly and without any further loss of life.

We are also following the events closely as Boston is the site of the Experimental Biology meeting that starts this weekend and runs into next week. According to the events website they are still planning on holding the meeting without any changes. They do, however, admit that since matters are changing rapidly that you should check their site often for updates and you will be notified if they decide to make any changes to the program as a result of breaking news.

Hopefully everything will be resolved and we will be able to see you all safely arrived in Boston when you stop by to see BMG LABTECH at booth 202.

Thursday, April 18, 2013

BMG LABTECH application note 206 describes the performance of the PHERAstar FS in detection of intracellular calcium and dopamine receptor activation. This is performed using two Invitrogen assays: The Fluo-4 Direct Calcium Assay and the Tango D1-bla U2OS GPCR Cellular Assay; which is based on arrestin recruitment to the dopamine receptor. The performance in each assay highlights the qualities of the PHERAstar FSthat make it such a fantastic HTS microplate reader.

Both assays use the bottom reading capabilities of the PHERAstar FS which is highly desirable for performance of cell based assays such as these. Furthermore the calcium assay exploits the injection at point of measurement capability of the PHERAstar FS which allows for precise measurement of fast kinetic assay such as this.

Finally, the GPCR assay employs the dual emission detection of the PHERAstar FSto assess whether FRET is occurring for substrate labeled with coumarin and fluorescein, which is indicative of inactivity of the receptor. Upon receptor activation beta-lactamase is expressed which cleaves the substrate leading to a decrease in FRET. Using the dual emission detection capabilities you can monitor the FRET emission at 530 nm and emission at 460 nm indicative of loss of FRET simultaneously.

Monday, April 15, 2013

20X magnification of the somatosensory cortex of a mouse brain slice. These little guys have green flourescent protein (GFP) from a jelly-fish expressed in a subset of their neurons. Layer V neuron cell bodies are the teardrop shaped things at the bottom and then the dendrite reaches up like a tree to then bifurcate near the top (pial surface).by Robert Cudmore

A group from Stanford have developed a technique called CLARITY which allows intact tissues to be embedded in a hydrogel matrix. The result is a fully assembled but optically transparent tissue that can go through multiple rounds of staining and destaining to reveal the inner structure of the brain. This approach alleviates the painstaking process of sectioning the brain which also has detrimental effects of the structures within the brain.

The procedure involves suspending the brain, or other tissue, in the hydrogel matrix which then forms a hybrid with the proteins, nucleic acids and small molecules found in the organ. The fats are excluded and can be dissolved away in an electric field and the fine structures of the brain can then be observed. This technique will hopefully reveal how different diseases affect brain structure.

Kinetic curves obtained from the M1/M17 fluorescent assay.
Figure is taken directly from the MARS data analysis software.

This application highlights the multimode capabilities of the FLUOstar Omega which allows the user to monitor both fluorescence and absorbance in the same well. As a result the effect of a single inhibitor on all three peptidases could assessed simultaneously. Fluorescent detection of the cleavage of methylcoumarin by M1/M17 could be measured at 335 nm excitation and 460 emission and absorbance at 405 nm is indicative of M18 cleavage of a nitroanalide substrate. In this way a 400 compound library was screened in a day.

Application note 235, now available on the BMG LABTECH website, describes the use of the multifunctional microplate reader the FLUOstar Omega to perform a screen for inhibitors of metalloaminopeptidases. Screening for these inhibitors will hopefully reveal new antimalarial agents.

Malaria is a mosquito-borne disease which results when a host organism is infected with the protozoan parasite Plasmodium vivax or other members of the genus Plasmodium. While the disease is restricted to the tropics it still accounts for nearly one million deaths per year. Therefore the search for new treatments is an area of intense research.

Malarial sporozites develop inside oocytes and are
released in large numbers into the hemocoel ofAnopheles stephensi mosquitoes. This false-colored
electron micrograph shows a sporozite migrating through
the cytoplasm of midgut epithelia.Image by Ute Frever, false color by Margaret Shear

During an infection metalloaminopeptidases digest host hemoglobin and are essential for parasite survival. So, finding inhibitors of these enzymes could result in novel therapeutic options.

As with all screening strategies it is important to decrease the amount of compound used. To this end the current application note details a technique that can monitor activity of three metalloaminopeptidases at the same time.

Using the multifunctional capabilities of the FLUOstar Omega from BMG LABTECH the breakdown of substrates was monitored using fluorescence and absorbance modes. Thus kinetic assessment of enzyme activity could be monitored and inhibitors could be screened.

The soup has many variations but is based on a salty beef and soy broth and contains sliced hard boiled egg both of which have been shown to ease hangovers.

Space-filling model the acetaldehyde molecule

The broth can help replace the salts like sodium and potassium that are lost due to the diuretic effect of alcohol. Furthermore, eggs contain cysteine, which helps remove acetaldehyde from the body. Acetaldehyde, which has toxic effects, is produced when the body breaks down alcohol.

'Old Sober' has been a popular remedy in New Orleans since the 1950's for those that have enjoyed the nightlife on Bourbon Street a bit too excessively.

Of course the only way to truly avoid a hangover is to not consume alcohol in the first place.

Tuesday, April 9, 2013

One way to maximize your inhibitor screens is to get more out of each treatment. For example if you can monitor the activity of multiple enzymes in the same well you will get more results from each individual volume of inhibitor.

Friday, April 5, 2013

BMG LABTECH will also attend the EuroLab 15th International Trade Fair of Analytical, Measurement and Control Technology. This event will be held on April 10-12 in Warsaw, Poland and features a combination science and business experts that make it an excellent source for information about current scientific techniques and technology.

For more information on these and other events at which BMG will attend please the Events Page on our website.

Thursday, April 4, 2013

One of the capabilities that all BMG LABTECH microplate readers
have is the script function which allows multiple functions to be performed
without the need for user intervention. An example of this capability is seen
in BMG application note 207 where absorbance was used to measure bacterial
growth and dual fluorescence intensity measurements were used to detect oxygen
consumption.

Comparison between O2 and OD600 profiles
(Multiplexed measurement)

Bacterial growth
was determined using OD600 while oxygen consumption was measured
using the MitoXpress® probe from Luxcel Biosciences. Thus multiparametric
analysis of cell growth could be performed to assess replication as determined
by OD600 as well as growth and alterations in metabolism determined
from oxygen consumption.

This sort of multiparametric analysis is useful when
assessing the affects of drug treatment and genetic alterations and the use of
the BMG LABTECHFLUOstar Omega allows this analysis to be performed with high
throughput.

The production of knock out mice is a labor intensive, expensive and time consuming process. However, these mice with altered genes or gene regions are invaluable to basic research into how diseases are triggered at the cell level. To traditionally produce a knock out mouse, scientists create stem cells that are genetically modified which are implanted into an embryo. The result is mice with both modified and unmodified cells that must be cross bred several times until the knockout characteristic is carried in all cells. In sum this process can take up to 2 years.

Now, a recent publication in PNAS reports the findings of a collaboration of German scientists that can produce a knockout mouse in under 5 months! They used transcription activator-like effector nuclease (TALEN) enzymes to directly modify genes in the fertilized mouse egg so that all cells in the mouse would carry the same genetic defect. Thus eliminating the breeding of numerous mice to eventually result in the desired knockout.

TALEN enzymes are dual functional; one part binds to a particular gene while another part cuts the DNA. Creation of variants in the binding function will allow scientists to make precise DNA cuts that can be used to modify specific genes.

Tuesday, April 2, 2013

Last Thursday BMG LABTECH participated in a webinar entitled: Improvements in Microplate Technology and Its Impact on Your Efficiency. The event was hosted by Lab Manager magazine and the recorded webinar can be viewed at their website:

Monday, April 1, 2013

BMG LABTECH’s PHERAstar
FS will be featured in the laboratory of CBS’ popular CSI Las Vegas television crime drama.

After having heard, and rightly so, that the PHERAstar
FS is the “Gold Standard” in
microplate readers, the prop master for CSI
contacted BMG LABTECH US and requested a unit to be used on the show. We were
happy to comply and sent a PHERAstar
FS, along with our US Southern California
Sales Manager, Lee Hedges, up to Hollywood.

The episode plot utilizes the PHERAstar
FS to perform an assay to determine
if a victim has a possible allergy. The assay used is a fluorescence
alternative to the RAST test (radioallergosorbent
test), which uses a blood sample to test for allergies to various substances. Ultimately,
the PHERAstar
FS was used by CSI lab
technician, David Hodges, in bringing a suspect to justice.

The PHERAstar FS on the set of CSI Las Vegas

On the PHERAstar
FS, prop master Michael
Lindsay enthused, “It is a really cool machine!” Thanks, Michael, we humbly agree!
The PHERAstar
FS is the Gold-Standard multidetection
microplate reader for HTS and assay development. It is easily integrated,
compact, versatile, and its sensitivity is unmatched.

Tune in to CSI on May 1, 10:00 pm ET/PT to watch the show. (Season
13, Episode 20) and make sure to look for the PHERAstar
FS in the lab scenes! The PHERAstar
FS is also slated to be used in
Season 14.